Excitation of central neurons by dieldrin and picrotoxinin in susceptible and resistant Drosophila melanogaster (meigen)

Neurophysiological studies were used to characterize the resistance mechanism in a new cyclodiene-resistant strain of Drosophila melanogaster. Suction electrode recordings were taken from the peripheral nerves of transected larval central nervous system. Treatment of nerve preparations with 1 mM GABA reduced the spontaneous firing of peripheral nerves. This inhibition was effectively reversed within 10 min by exposing preparations from susceptible insects (Oregon-R wild type) to 10 μM dieldrin. In contrast, 30 min incubations with 10 μM dieldrin had no effect on preparations from resistant individuals. At 10 μM, picrotoxinin was also effective in antagonizing the action of GABA in susceptible nerve preparations. In recordings from resistant insects (n = 3), picrotoxinin displayed either no antagonism of GABA-dependent inhibition, weak antagonism of GABA, or hyperexcitation indistinguishable from those of susceptible preparations. These results demonstrate that cyclodiene resistance in the Maryland strain of D. melanogaster is present at the level of the nerve, and that the resistance extends to picrotoxinin, albeit at a reduced level. The possible role of an altered GABA receptor in this resistance is discussed.     

Mode of action of the plant-derived silphinenes on insect and mammalian GABAA receptor/chloride channel complex

The silphinenes are tricyclic sesquiterpenes that have antifeedant and toxic effects in insects and structural similarity to the known GABA antagonist, picrotoxinin. In murine synaptoneurosomes, silphinenes block GABA-stimulated influx of ³⁶Cl⁻ with EC₅₀s in the range of 10-30 μM. In insects, silphinenes were tested in neurophysiological recordings of central neurons from third instar Drosophila melanogaster larvae. Silphinenes reversed the blockage of neuronal firing induced by GABA, but had little effect below 100 μM. The structure-activity profile observed in the murine chloride flux assay was also observed in the larval neurophysiological assay, indicating little selectivity for the silphinenes. A reference silphinene was equally active on nerve preparations from the rdl strain of D. melanogaster, which is resistant to channel-blocking antagonists via an altered GABA receptor. This latter finding suggests that silphinenes interact with the insect GABA receptor in a manner somewhat different from PTX, and that rdl resistance in the field may have little effect on silphinene efficacy.     

Spirosultam LY219048: A new chemical class of insecticide acting upon the GABA receptor/chloride ionophore complex

This study assessed the toxicity and mode of action of a new experimental insecticide, LY219048 in insects and mammals. LY219048 produced rapid convulsions in mice and had LD₅₀ values of 0.7 mg kg^?1 and 4 mg kg^?1 after intracerebral and intraperitoneal injection, respectively. In initial screens against insects, LY219048 showed low activity against the German cockroach (Blatella germanica L.). Lethality from dietary exposure required one to two weeks, even at concentrations as high as 10000 mg kg^?1 (LC₅₀ = 485 mg kg^?1). In contrast, it had an LC₅₀ value of 8.3 mg kg^?1 against insecticide-susceptible Drosophila melanogaster (Meig.) when synergized with piperonyl butoxide. Significant resistance to LY219048 (> 12-fold) was detected in a cyclodiene-resistant strain of D. melanogaster possessing an altered target site resistance mechanism. This finding suggested that LY219048 blocked the 4-aminobutyric acid (GABA)-gated chloride channel in a manner similar to that of the cyclodienes. In physiological studies in larval D. melanogaster central neurons, LY219048 antagonized the reduction of firing caused by 1 mM GABA. Dose-response experiments showed that the ED₅₀ for blocking inhibition under these conditions was c. 1 μ. Studies of ³⁶CI uptake into bovine brain synaptosomes found that LY219048 was a potent antagonist. At 10 μ it completely blocked chloride flux stimulated by 50 μM GABA. LY219048 competitively displaced [³H]TBOB binding from bovine brain membranes, with an IC₅₀ of 42 nM, which was comparable to values determined for TBPS (35 nM) and picrotoxinin (267 nM). There was little or no displacement (<25%) of [³H]flunitrazepam or [³H]muscimol binding by 10 μM LY219048. Taken together, these results provide strong evidence that this new chemical class of insecticide manifests its acute toxicity by blocking the GABA-gated chloride channel.     

Quantitative structure-activity relationships of monoterpenoid binding activities to the housefly GABA receptor

BACKGROUND: Monoterpenoids are a large group of plant secondary metabolites. Many of these naturally occurring compounds have shown good insecticidal potency on pest insects. Previous studies in this laboratory have indicated that some monoterpenoids have positive modulatory effects on insect GABA receptors. In this study, the key properties of monoterpenoids involved in monoterpenoid binding activity at the housefly GABA receptor were determined by developing quantitative structure‐activity relationship (QSAR) models, and the relationship between the toxicities of these monoterpenoids and their GABA receptor binding activities was evaluated. RESULTS: Two QSAR models were determined for nine monoterpenoids showing significant effects on [³H]‐TBOB binding and for nine p‐menthane analogs with at least one oxygen atom attached to the ring. The Mulliken charges on certain carbon atoms, the log P value and the total energy showed significant relationships with binding activities to the housefly GABA receptor in these two QSAR models. CONCLUSIONS: From the QSAR models, some chemical and structural parameters, including the electronic properties, hydrophobicity and stability of monoterpenoid molecules, were suggested to be strongly involved in binding activities to the housefly GABA receptor. These findings will help to understand the mode of action of these natural insecticides, and provide guidance to predict more monoterpenoid insecticides. Copyright © 2012 Society of Chemical Industry     

Detection of cyclodiene insecticide resistance-associated mutations by single-stranded conformational polymorphism analysis

Cyclodiene insecticide resistance is associated with replacements of a single amino acid within the putative lining of a δ-aminobutyric acid (GABA)-gated chloride ion channel gene Resistance to dieldrin (Rdl). Only two resistance-associated amino acid replacements have been identified; alanine to serine in Drosophila melanogaster, D. simulans, Aedes aegypti, and Tribolium castaneum and alanine to glycine as a second allele in D. simulans. Here we report that single stranded conformational polymorphism (SSCP) analysis of genomic DNA, amplified by the polymerase chain reaction (PCR) for exon 7 of the Rdl gene, can be used to genotype strains or individuals of all of these insects. This technique also appears simultaneously to distinguish between D. melanogaster and D. simulans, sibling species only reliably identifiable by examination of male genitalia. The relative advantages of this genotyping technique against other PCR-mediated techniques in monitoring for insecticide resistance are discussed.     

Difference in the picrotoxinin receptor between the cyclodiene-resistant and susceptible strains of the german cockroach

As a result of toxicity tests, it was established that all cyclodiene-resistant strains of the German cockroach are also resistant to picrotoxinin, a plant-origin neurotoxicant. Two of the cockroach strains which exhibit a distinct cross-resistance pattern to picrotoxinin (i.e., LPP and FRP) are the ones that have been purified genetically by backcrossing against the susceptible (CSMA) strain. This cross-resistance pattern appears to be specific to picrotoxinin and does not extend to other neuroexcitants such as bicuculline, beta-bungarotoxin, and DDT. The nervous system of the resistant cockroach was found to be less sensitive to picrotoxinin. Furthermore, it was determined that nerve components from the resistant cockroaches have significantly lower binding capacity to [³H]α-dihydropicrotoxinin. The most likely explanation for the above phenomenon is that these cockroaches have developed the cyclodiene resistance by altering the nerve receptor for picrotoxinin.     

Picrotoxinin receptor in the central nervous system of the American cockroach: Its role in the action of cyclodiene-type insecticides

The nature of the picrotoxinin receptor was studied using the central nervous system (CNS) of the American cockroach. It first was confirmed by using an electrophysiological technique that the abdominal nerve cord of the American cockroach was sensitive to picrotoxinin. By using a $\text{[}{}^3\text{H]α-dihydropicrotoxinin}$ binding test it was determined that the picrotoxinin receptor in CNS of this insect had a higher affinity toward picrotoxinin and heptachlor epoxide than the corresponding receptor in the rat brain. Also, the cockroach brain preparation had a higher percentage of specific binding in the total binding, making this material suitable for receptor studies. By using a sucrose density centrifugation technique, it was determined that the fraction sedimented at the interphase of 1.0 to 1.2 M sucrose at 100,000g contained the highest level of specific binding site. The receptor showed a sensitivity to all insecticidal cyclodienes tested, namely photodieldrin, oxychlordane, endrin, heptachlor epoxide, γ-chlordane, dieldrin, aldrin, heptachlor, and isodrin (expressed in the order of potency). Among four BHC isomers, the γ-isomer showed the highest potency to bind with this receptor.     

The role of GABA and glutamate receptors in susceptibility and resistance to chloride channel blocker insecticides

Despite a point mutation in the pore-forming segment of the Rdl GABA receptor subunit that is widespread and persistent in insect populations and confers high levels of resistance to dieldrin and other polychlorocycloalkane (PCCA) insecticides, the phenylpyrazole insecticide fipronil, which binds at same site, has proven to be effective in controlling many insects, including dieldrin-resistant populations. Fipronil and its major sulfone metabolite are unique among chloride channel blocking insecticides in that they also potently block GluCls. We present here a patch clamp study of the action of fipronil sulfone on native GABA receptors and GluCl receptors from susceptible and dieldrin-resistant German cockroaches, to provide a better understanding of the effect of the Rdl mutation on the function and insecticide sensitivity of these two targets, and its role in resistance. Dieldrin blocked GABA currents with an IC₅₀ of 3 nM in wild-type cockroaches, and 383 nM in resistant insects, yielding a resistance ratio of 128. Fipronil sulfone blocked GABA currents with an IC₅₀ of 0.8 nM in susceptible insects and 12.1 nM, or 15-fold higher, in resistant insects. While both GluClD (desensitizing) and GluClN (non-desensitizing) receptors were found in German cockroach neurons, GluClN receptors were rare and could not be included in this study. GluClD receptors from resistant insects had reduced sensitivity to glutamate and a lower rate of desensitization than those from susceptible insects, but their sensitivity to block by fipronil sulfone was not significantly changed, with an IC₅₀ of 38.5 ± 2.4 nM (n = 8) in the susceptible strain and 40.3 ± 1.0 nM (n = 7) in the resistant strain. Fipronil sulfone also slowed the decay time course of GluClD currents. These results suggest that GluClD receptors contain the Rdl subunit, but their sensitivity to fipronil sulfone is not altered in resistant insects.     

GABA受体的分离纯化方法研究进展

γ-氨基丁酸(gamma- aminobutyric acid,GABA)受体是昆虫体内重要的抑制性神经递质受体,是多种杀虫剂的重要作用靶标,其结构与相关农药的毒理性质和害虫的抗药性密切相关。对昆虫GABA受体进行分离纯化并研究其立体结构,对于研究开发高效、低毒、安全的新农药及克服害虫抗药性均具有重要的指导意义。GABA受体的分离、纯化方法与其分子质量大小、带电性和特异性亲和力有关。对GABA受体的主要分离纯化方法:沉淀法、密度梯度离心法、离子交换色谱法、羟基磷灰石色谱法、凝胶过滤法和亲和色谱法等进行了综述,评述了不同纯化方法的优缺点,并总结了分离所用各种去污剂对该受体提取效率的影响。     

A native picrotoxin-resistant GABA-gated chloride channel receptor subtype in cockroach neurons

Among insect GABA receptors, the GABA-gated chloride channel subtype is insensitive to bicuculline and has been thought to be composed of two populations because of differences in chloride conductance increase, GABA and picrotoxin (PTX) sensitivity. To characterize this possible diversity in GABA-gated chloride channels, electropharmacological experiments were performed on giant interneuron synaptic GABA receptors and on somatic GABA receptors of dorsal unpaired median (DUM) neuron and fast coxal depressor (D_f) motoneuron of the cockroach Periplaneta americana (L). Electrophysiological assays performed at cercal-afferent giant interneuron synapses demonstrated that a biphasic increase in membrane conductance, in response to long-lasting (30 s) neuropilar microapplication of GABA, could be explained by the existence of two GABA-operated chloride channel receptor subtypes. The low stable membrane conductance increase, representing less than 30% of the maximum reached during the early transient phase, was not desensitized quickly. It was reproduced by neuropilar microapplication of cis-4-aminocrotonic acid (CACA) and, in contrast to the fast phase, was not antagonized by bath application of 10⁻⁵ M PTX. Long-lasting (3 min) pneumatic pressure application of GABA on the cell body of motoneuron D_f evoked a fast transient hyperpolarization followed by a slower phase of further hyperpolarization. PTX (10⁻⁵ M ) blocked the fast transient phase and revealed a slow stable hyperpolarization. PTX (10⁻⁴ M ) blocked the major part of the remaining GABA response. The slow hyperpolarization was reproduced by application of CACA. Similar effects of GABA and CACA were recorded on DUM neuron cell bodies. All of these observations are consistent with the possible existence of two GABA-gated chloride channel subtypes in the insect CNS. © 1999 Society of Chemical Industry     

Action of avermectin B1a on the leg muscles and the nervous system of the American cockroach

The action of avermectin was studied in the leg muscle and the central nervous system of the American cockroach, Periplanata americana L. Avermectin at a low concentration (10^?7M) causes a failure of the leg muscles to respond to external stimuli within 30 min without affecting the magnitude of contraction. Avermectin was found to stimulate Cl^? uptake by the leg muscles within 4 min at 10^?7M. The threshold concentration to cause such stimulation was on the order of 10^?8M. This stimulatory action could be antagonized by picrotoxinin (10^?4M) and to a lesser extent by bicuculline methiodide (10^?4M). The phenomenon is observable under both Na⁺-free and K⁺-free conditions. It was concluded that the action of avermectin is to open the chloride channel on the plasma membrane. This action of avermectin does not seem to be mediated through GABA, GABA receptors, diazepine receptors, or picrotoxinin receptor in this insect species, and therefore suggests that avermectin directly attacks the chloride channel proper both in the central nervous and the neuromuscular systems.     

Mutation of the GABA receptor associated with fipronil resistance in the whitebacked planthopper, Sogatella furcifera

We found the A2′N mutation (index number for M2 membrane spanning region) in the GABA receptor subunit of fipronil-resistant Sogatella furcifera, by analyzing DNA sequences amplified from fipronil-resistant and -susceptible S. furcifera. In order to confirm the role of A2′N mutation in the fipronil resistance, we expressed the wild-type and A2′N mutant Drosophila GABA receptors in Drosophila Mel-2 cells stably. Amino acid sequences of three membrane spanning regions (M1-M3), which are important for binding of fipronil, are conserved between Drosophila and S. furcifera. So the results of A2′N mutant Drosophila GABA receptor suggest the role of A2′N mutation in fipronil-resistant S. furcifera. The membrane potential assay showed that the A2′N mutant Drosophila GABA receptor was not inhibited by fipronil at all, while the IC₅₀ value of fipronil for wild-type Drosophila GABA receptor was 172 nM. These results suggest that A2′N mutation confers the resistance of fipronil in S. furcifera.     

Bicyclophosphorothionate antagonists exhibiting selectivity for housefly GABA receptors

2,6,7-Trioxa-1-phosphabicyclo[2.2.2]octane 1-sulfides (bicyclophosphorothionates) with various C_1–4 alkyl groups at the 3- and 4-positions were synthesized and tested for their ability to compete with [³H]4′-ethynyl-4-n-propylbicycloorthobenzoate (EBOB), a non-competitive antagonist of γ-aminobutyric acid (GABA) receptors, for specific binding to rat-brain and housefly-head membranes, and for their insecticidal activity against houseflies. Among the 3,4-substituted analogues, 20 compounds were selectively active for housefly GABA receptors versus rat GABA receptors. The 3-alkyl groups of C₃ length and the 4-alkyl groups of C₄ length were tolerated in housefly receptors, whereas such bulky substituents were deleterious in rat receptors. The 4-isobutyl-3-isopropyl analogue was the most potent in housefly receptors (IC₅₀ = 45.2 nM ), and tert-butylbicyclophosphorothionate (TBPS), with the 4-tert-butyl group and no 3-substituent, was the most potent in rat receptors (IC₅₀ = 62.2 nM ). Their receptor selectivities (rat IC₅₀/housefly IC₅₀) were 52 and 0.038, respectively. The insecticidal activity (LD₅₀) of 20 active analogues was well correlated with their potency (IC₅₀) in inhibiting [³H]EBOB binding to housefly-head membranes (r = 0.93). The results obtained in the present study indicate that the introduction of appropriate alkyl groups into the 3- and 4-positions of bicyclophosphorothionate leads to non-competitive antagonists with increased affinity and selectivity for housefly ionotropic GABA receptors versus rat GABA_A receptors. © 1999 Society of Chemical Industry     

Quantitative autoradiography of GABA receptors in locust (Schistocerca americana) brain

Conventional film autoradiography was used at the light microscopic level for the localization and quantization of 4-aminobutyric acid (GABA) receptors in the locust brain (Schistocerca americana). Localization of the receptor site was achieved via binding with the receptor-ligand probe [³ H]muscimol. Frozen sections were cut and subsequently incubated either in 40 nM [³H]muscimol or by coincubating sections with [³H]muscimol and one of the following: GABA (50 μM)], a receptor specific agonist [muscimol (1 μM) or isoguvacine (1 μM)], an uptake inhibitor [nipecotic acid (50 μM)], or a noncompetitive channel modulator [avermectin B_1a, (1 μM) or aldrin (50 μM)]. Through computer image enhancement and densitometric analysis of the optical density of [³H]muscimol binding sites, the interaction of the above compounds with the putative GABA receptor was determined for various anatomical regions of the locust brain. By comparing the differently treated, but adjacent sections, GABA receptor distribution was quantitated and mapped. Receptor sites were found distributed in the antennal lobes, central body, β-lobe and β-lobe of the corpus pedunculatum, protocerebral bridge, and calyx as well as the optic lobe regions.     

Effects of the essential oil constituent thymol and other neuroactive chemicals on flight motor activity and wing beat frequency in the blowfly Phaenicia sericata

BACKGROUND: The effects were evaluated of the plant terpenoid thymol and eight other neuroactive compounds on flight muscle impulses (FMIs) and wing beat frequency (WBF) of tethered blowflies (Phaenicia sericata Meig.). RESULTS: The electrical activity of the dorsolongitudinal flight muscles was closely linked to the WBF of control insects. Topically applied thymol inhibited WBF within 15–30 min and reduced FMI frequency. Octopamine and chlordimeform caused a similar, early‐onset bursting pattern that decreased in amplitude with time. Desmethylchlordimeform blocked wing beating within 60 min and generated a profile of continuous but lower‐frequency FMIs. Fipronil suppressed wing beating and induced a pattern of continuous, variable‐frequency spiking that diminished gradually over 6 h. Cypermethrin‐ and rotenone‐treated flies had initial strong FMIs that declined with time. In flies injected with GABA, the FMIs were generally unidirectional and frequency was reduced, as was seen with thymol. CONCLUSIONS: Thymol readily penetrates the cuticle and interferes with flight muscle and central nervous function in the blowfly. The similarity of the action of thymol and GABA suggests that this terpenoid acts centrally in blowflies by mimicking or facilitating GABA action. Copyright © 2009 Society of Chemical Industry     

Insecticidal activity of Sikimi extract and its modulation of the GABAA receptor channel

Sikimi plant (also known as Japanese star anise), Illicium anisatium, is toxic to mammals. Extracts of Sikimi were studied for their insecticidal activity against the larvae of mosquito, Culex quinquefasciatus, and for their mechanism of action on ion channels. Crude methanol extract and its ethyl acetate-soluble fractions were insecticidally active, with EC₅₀ values of 63·0 μg ml^-1 and 43·7 μg ml^-1, respectively. The ethyl acetate-soluble fraction was perfused through the bathing solution and the current induced by a brief (10 ms) application of GABA by pressure ejection through pipette electrode was recorded by the whole-cell patch clamp technique. The extract suppressed GABA-induced currents irreversibly with an EC₅₀ value of 0·42 μg ml^-1. The time constant of current fitted to the single exponential function was shortened by the ethyl acetate-soluble fraction at concentrations ranging from 0·1 μg ml^-1 to 10 μg ml^-1 in a concentration-dependent manner. It was concluded that Sikimi extracts decreased the affinity of GABA for its binding site on the GABA receptor, thereby suppressing GABA-induced currents. © 1998 SCI     

Biochemical characteristics of insecticide resistance in the fall armyworm, Spodoptera frugiperda (J.E. Smith)

A strain of the fall armyworm, Spodoptera frugiperda (J.E. Smith), collected from corn in Citra, Florida, showed high resistance to carbaryl (562-fold) and methyl parathion (354-fold). Biochemical studies revealed that various detoxification enzyme activities were higher in the field strain than in the susceptible strain. In larval midguts, activities of microsomal oxidases (epoxidases, hydroxylase, sulfoxidase, N-demethylase, and O-demethylase) and hydrolases (general esterase, carboxylesterase, β-glucosidase) were 1.2- to 1.9-fold higher in the field strain than in the susceptible strain. In larval fat bodies, various activities of microsomal oxidases (epoxidases, hydroxylase, N-demethylase, O-demethylases, and S-demethylase), glutathione S-transferases (CDNB, DCNB, and p-nitrophenyl acetate conjugation), hydrolases (general esterase, carboxylesterase, β-glucosidase, and carboxylamidase) and reductases (juglone reductase and cytochrome c reductase) were 1.3- to 7.7-fold higher in the field strain than in the susceptible strain. Cytochrome P450 level was 2.5-fold higher in the field strain than in the susceptible strain. In adult abdomens, their detoxification enzyme activities were generally lower than those in larval midguts or fat bodies; this is especially true when microsomal oxidases are considered. However, activities of microsomal oxidases (S-demethylase), hydrolases (general esterase and permethrin esterase) and reductases (juglone reductase and cytochrome c reductase) were 1.5- to 3.0-fold higher in the field strain than in the susceptible strain. Levels of cytochrome P450 and cytochrome b₅ were 2.1 and 1.9-fold higher, respectively, in the field strain than in the susceptible strain. In addition, acetylcholinesterase from the field strain was 2- to 85-fold less sensitive than that from the susceptible strain to inhibition by carbamates (carbaryl, propoxur, carbofuran, bendiocarb, thiodicarb) and organophosphates (methyl paraoxon, paraoxon, dichlorvos), insensitivity being highest toward carbaryl. Kinetics studies showed that the apparent K_m value for acetylcholinesterase from the field strain was 56% of that from the susceptible strain. The results indicated that the insecticide resistance observed in the field strain was due to multiple resistance mechanisms, including increased detoxification of these insecticides by microsomal oxidases, glutathione S-transferases, hydrolases and reductases, and target site insensitivity such as insensitive acetylcholinesterase. Resistance appeared to be correlated better with detoxification enzyme activities in larval fat bodies than in larval midguts, suggesting that the larval fat body is an ideal tissue source for comparing detoxification capability between insecticide-susceptible and -resistant insects.     

Effect of abamectin on electrophysiological activity in the ventral nerve cord of Periplaneta americana L.: Correlation with symptoms of poisoning and levels of toxicant in vivo

Twenty-four hours after injection of adult male Periplaneta americana with 36 ng [5-³H]abamectin, when symptoms of poisoning were apparent in most individuals, similar levels of radioactivity were found in tissue samples from the ventral nerve cord, metathoracic muscle, fat body and testis of randomly-selected insects. When insects injected with [5-³H]abamectin (36 ng) were separated after 24 h into three groups showing varying symptoms, the level of radioactivity in the ventral nerve cord was found to be significantly greater in partially-paralysed and paralysed groups (4- and 7-fold respectively) than in a treated but non- paralysed group. The mean levels of abamectin in the nerve cords of unaffected, ‘partially-paralysed’ and ‘paralysed’ insects were estimated to be 4, 23 and 37 nM respectively. Extracellular studies of the in-vitro action of abamectin (10 nM and 1 μM) on the spontaneous activity of a picrotoxin-sensitive, partially desheathed, ventral nerve cord preparation of adult male P. americana found the mean response time to be relatively slow (77 and 38 min respectively). In sheathed nerve cords, the mean response time to abamectin (1 μM) was 117 min. In all cases, treatment with abamectin increased the stimulus voltage required to evoke a response. Spontaneous activity was found to be progressively reduced in ventral nerve cord preparations from ‘partially-paralysed’ and ‘paralysed’ insects compared with ‘non-paralysed’ and untreated insects following dissection 24 h after injection of P. americana with unlabelled or labelled abamectin (36 ng), while the stimulus required to evoke activity in the ventral nerve cord showed the reverse trend. These differences in electrophysiological activity could be correlated directly with varying levels of abamectin in the nerve cords of ‘paralysed’ and ‘partially-paralysed’ insects.     

Glutamate-activated chloride channels: Unique fipronil targets present in insects but not in mammals

Selectivity to insects over mammals is one of the important characteristics for a chemical to become a useful insecticide. Fipronil was found to block cockroach GABA receptors more potently than rat GABA_A receptors. Furthermore, glutamate-activated chloride channels (GluCls), which are present in cockroaches but not in mammals, were very sensitive to the blocking action of fipronil. The IC₅₀s of fipronil block were 30 nM in cockroach GABA receptors and 1600 nM in rat GABA_A receptors. Moreover, GluCls of cockroach neurons had low IC₅₀s for fipronil. Two types of glutamate-induced chloride current were observed: desensitizing and non-desensitizing, with fipronil IC₅₀s of 800 and 10 nM, respectively. We have developed methods to separately record these two types of GluCls. The non-desensitizing and desensitizing currents were selectively inhibited by trypsin and polyvinylpyrrolidone, respectively. In conclusion, in addition to GABA receptors, GluCls play a crucial role in selectivity of fipronil to insects over mammals. GluCls form the basis for development of selective and safe insecticides.